Neurotoxigenic strains of Clostridium butyricum and Clostridium baratii produce botulinum neurotoxins (BoNTs), which are the most potent neurotoxins known. BoNTs are characterized as Category A Select Agents and are considered potential bioterrorism threats (Arnon, S. S. et al. 2001, Jama 285:1059-1070).
BoNT is synthesized as a single chain polypeptide (molecular weight of 150 kDa) with relatively little toxic potency. It becomes toxic upon cleavage by trypsin or bacterial enzymes into a heavy chain (100 kDa) and a light chain (50 kDa). Three dimensional structure shows that BoNTs contain a receptor-binding domain located in the C-terminal region of the heavy chain, a catalytic domain (the light chain) with endopeptidase activity on neuronal substrates and a translocation domain located in the N-terminal region of the heavy chain.
BoNTs can be immunologically distinguished using homologous antitoxins into seven serotypes, designated A-G. Different serotypes of BoNTs have regions of homology, particularly in the residues defining the catalytic active site, in the translocation domain, and in the two cysteine residues forming the disulfide bond connecting the heavy chain and the light chain. The least degree of homology is in the carboxyl region of the heavy chain, which is involved in neurospecific binding. Indeed, among these serotype distinctions there is considerable genetic variation, as demonstrated by the recognition of at least 24 subtypes (Carter, A. T., et al., 2009, BMC Genomics 10:115; Dover, N., et al., 2009, J. Clin. Microbiol. 47:2349-2350; Hill, K. K. et al. 2007, J. Bacteriol 189:818-832; Smith, T. J. et al. 2005, Infect. Immun. 73:5450-5457). These subtypes have been distinguished based on their degree of genetic variation with subtypes having a minimum of 2.6% divergence on the amino acid level (Webb, R. P., et al., 2009, Vaccine 27:4490-4497). These subtypes can also be distinguished by mouse bioassay, as a new subtype is resistant to neutralization by antibodies raised against known subtypes.
Despite of the variations, different serotypes of BoNTs act through a similar mechanism: by inhibiting the release of acetylcholine, a neurotransmitter, from the presynaptic nerve terminal and thus causing local chemodenervation. The action of BoNTs involves a four step process: (1) high affinity, serotype specific binding by the heavy chains to receptors on presynaptic membrane of cholinergic nerve endings; (2) receptor mediated, energy dependent internalization of the complex; (3) translocation from the acidic endosome to the cytosol; and (4) enzymatic cleavage, by the light chain, of specific proteins that are critical for fusion of the presynaptic acetylcholine vesicle with the presynaptic membrane, thus preventing release of acetylcholine into the synapse.
BoNT/A is of particular importance and interest since it is the most significant threat in bioterrorism and has been increasingly used as a pharmaceutical modality (Aoki, K. R. 2003, Clin. Dermatol. 21:476-480; Delgado, M. R. 2003, J. Am. Acad. Orthop. Surg. 11:291-294). Thus far, four distinct subtypes of BoNT type A, i.e., BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4, have been identified in this manner (Arndt, J. W. et al. 2006, J. Mol. Biol. 362:733-742; Smith, T. J. et al. 2007, PLoS ONE 2:e1271).
The success of BoNTs as a therapeutic derives from certain important attributes of the toxin: (a) exceptionally specific binding to the presynaptic membrane of cholinergic terminals; (b) extremely high potency; (c) remarkable specificity for catalytic cleavage of proteins involved in neurotransmitter trafficking and exocytosis; (d) minimal spread from the injection site; (e) limited and mild adverse effects, and (f) extraordinary long duration of action. Table 1 summarizes the clinical applications of BoNTs.
Although each injection of BoNT has a long duration of effective action, repeated injection is necessary at about three month intervals because although the affected nerve terminals are no longer capable of neurotransmitter exocytosis, newly formed sprouts do release acetylcholine and form a functional synapse. As a result, after about three months, the original terminal resumes exocytosis and the sprouts regress to return the neuromuscular junction to its original state.
TABLE 1Clinical applications of botulinum toxinDystoniaBlepharospasm and lid apraxiaOromandibular-facial-lingual dystoniaCervical dystonia (torticollis)Laryngeal dystonia (spasmodic dysphonia)Limb dystoniaTask specific dystonia (eg, writer's or other occupational cramps)Other focal/segmental dystonias (primary, secondary)Other involuntary movementsHemifacial spasmLimb, head, voice, chin tremorPalatal myoclonusMotor and phonic tics (including coprolalia)Nystagmus and oscillopsiaMyokymiaInappropriate muscle contractionsSpasticity (stroke, cerebral palsy, head injury, multiple sclerosis)Painful rigidityStrabismusBruxism and temporo-mandibular joint syndromeStutteringChronic tension (muscle contraction) headachesLumbosacral strain and back spasmsRadiculopathy with secondary muscle spasmMyofascial pain syndromesAchalasia (lower oesophageal sphincter spasm)Spasm of the inferior constrictor of the pharynxSpasm of the sphincter of OddiSpastic bladder, detrusor sphincter dyssynergiaAnismusVaginismusOther applicationsProtective ptosisHyperlachrymationDrooling (sialorrhoea)HyperhidrosisGustatory sweatingAnal fissureConstipationObesity (distal stomach)Cosmetic (wrinkles, brow furrows, frown lines, “crow's feet”, platysma lines, facial asymmetry)Tennis elbow and other sports injuries
A growing impediment in BoNT administration is the development in patients of antibodies that react with and neutralize the toxin, thereby eliminating the effectiveness of the toxin for medicinal and cosmetic purposes. As a result, some patients become unresponsive to subsequent repeated treatments. Studies have shown that the heavy chain of the protein, used for substrate binding, is the primary portion against which humans develop antibodies against BoNT/A1, the predominat form of botulinum toxin used clinically.
The observation that BoNTs are chimeric molecules comprised of distinct protein domains suggests that designed chimeric neurotoxin could be constructed with enhanced or distinct therapeutic utility.
Needed in the art are novel subtypes of BoNTs which are not easily neutralized by existing antibodies in patients and thus possess distinct therapeutic utility, as well as novel subtypes of BoNTs which cause no or less development of antibodies in patients.